Simultaneous Determination of Tramadol and Acetaminophen in Human Plasma by LC–ESI–MS

This study presents a high-performance liquid chromatography–electrospray ionization–mass spectrometric (LC–ESI–MS) method for the simultaneous determination of tramadol and acetaminophen in human plasma using phenacetinum as the internal standard. After alkalization with saturated sodium bicarbonate, both compounds were extracted from human plasma with ethyl acetate and were separated by HPLC on a Hanbon LiChrospher CN column with a mobile phase of 10 mM ammonium acetate buffer containing 0.5% formic acid–methanol (40:60, v/v) at a flow rate of 1 mL min⁻¹. Analytes were determined using electrospray ionization in a single quadrupole mass spectrometer. LC–ESI–MS was performed in the positive selected-ion monitoring (SIM) mode using target ions at M+H]⁺ m/z 264.3 for tramadol, M+H]⁺ m/z 152.2 for acetaminophen and M+H]⁺ m/z 180.2 for phenacetinum. Calibration curves were linear over the range of 5–600 ng mL⁻¹ for tramadol and 0.03–16 μg mL⁻¹ for acetaminophen. The inter-run relative standard deviations were less than 14.4% for tramadol and 12.3% for acetaminophen. The intra-run relative standard deviations were less than 9.3% for tramadol and 7.9% for acetaminophen. The mean plasma extraction recovery for tramadol and acetaminophen were in the ranges of 82.7–85.9 and 83.6–85.3%. The method was applied to study the pharmacokinetics of a new formulation of tramadol/acetaminophen tablet in healthy Chinese volunteers.